Coagulation Factor IX
The blood coagulation factor IX (F.IX) plays a central role in the coagulation cascade. F.IX is a trypsin-like vitamin K-dependent serine protease that circulates in the plasma as a single chain inactive zymogen (DiScipio et al., 1977; Davie et al., 1991). Factor IX is activated by either factor XIa or by factor VIIa-tissue factor in a Ca2+ dependent manner. The activation requires cleavage of two peptide bonds by either the activated factor VII (F.VIIa)-tissue factor complex or activated factor XI (F.XIa) (Fujikawa et al., 1974; Lindquist et al., 1978) to remove a 35-residue activation peptide.
F.IX is a multi-domain protein. An N-terminal γ-carboxy glutamic acid (GLA) domain is followed by two epidermal growth factor-like (EGF) repeats, the activation peptide (AP) and a C-terminal serine protease domain with a trypsin-like active site (DiScipio et al., 1978). This domain structure defines the serine protease family of clotting factors (Furie and Furie, 1988), including also factor II (F.II), factor VII (F.VII), factor X (F.X), factor XI (F.XI), factor XII (F.XII), and protein C (PC). Within this family, F.IXa has unique proteolytic properties. Complex formation of F.IXa with F.VIIIa on a phospholipid surface increases reactivity against the natural substrate F.X 106-fold (Duffy and Lollar, 1992), while virtually no cleavage of peptides with corresponding F.X sequences was observed (McRae et al., 1981).
Freedman et al. (1996) proposed that the membrane binding site of factor IX resides in amino acid residues 1-11. In particular leucine 6, phenylalanine 9 and valine 10 were identified to form a hydrophobic site on the exterior of the FIX protein which buries inside the lipid bilayer.
Chang et al. (2002) describe that residues 102-108 in the EGF2-like domain of F.IX are important for proper binding to F.X. Wilkinson et al. obtained similar results for residues 88-109 (but not Arg94) and proposed their importance for assembly of the F.X activating complex on phospholipid vesicles or platelets (Wilkinson et al., 2002-a, Wilkinson et al., 2002-b).
Activated factor IX (F.IXa) then activates factor X (F.X) in a reaction that is dependent on the presence of calcium ions, a membrane surface (phospholipid), and a nonenzymatic protein cofactor, activated factor VIII (F.VIIIa) (Davie et al., 1991).
The importance of F.IXa in hemostasis is reflected by the occurrence of the bleeding disorder hemophilia B in individuals carrying mutations in the F.IX gene (Gianelli et al., 1998). F.IXa displays only very little proteolytic activity against natural or synthetic substrates in the absence of its cofactor F.VIIIa. Binding of F.VIIIa results in a 106-fold increase in proteolytic activity for F.X, whereas the activity with peptidic substrates remains less affected (Duffy and Lollar, 1992; McRae et al., 1981). The latter substrate-dependent activity of F.IXa modulation is similarly observed for the related coagulation enzymes activated PC (co-factor Protein S), F.Xa (co-factor Factor Va), F.VIIa (cofactor tissue factor), and FIIa (co-factor thrombomodulin), which in the presence of their cofactors, achieve a significant activity or specificity change with their natural substrates. (Mann et al. 2003). All coagulation serine proteases share extensive structural and functional homology.
Furthermore, the coagulation factors IXa (F.IXa) and Xa (F.Xa) both cleave natural substrates effectively only with a cofactor at a phospholipid surface. Hopfner et al. (1997) investigated variants of truncated F.IXa (rf9a) and F.Xa (rf10a) in E. coli to identify determinants of the difference in the amidolytic activity of F.IXa which is 104-fold lower than that of F.Xa. Based on the crystal structures of F.IXa and F.Xa four characteristic active site components (namely Glu219, the 148-loop, Ile213, the 99-loop, based on chymotrypsin numbering) were subsequently exchanged between rf9a and rf10a. Furthermore, combining all four mutations essentially introduced F.Xa properties into rf9a, i.e. the amidolytic activity was increased 130-fold with F.Xa substrate selectivity.
Enzymatically, F.IXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (F.VIIIa), distinguishing F.IXa from all other coagulation factors. Activation of the F.IXa-F.VIIIa complex requires its macromolecular substrate, factor X (F.X). The 99-loop positioned near the active site partly accounts for the poor activity of F.IXa because it adopts a conformation that interferes with canonical substrate binding in the subsites S2-S4. Sichler et al. (2003) disclose that residues Lys-98 and Tyr-99 (chymotrypsin numbering) are critically linked to the amidolytic properties of F.IXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 (rf9-Y94F/K98T/Y177T, chymotrypsin numbering)) produced an enzyme with 7000-fold increased activity and altered specificity towards factor Xa. Sichler et al. (2003) concluded, that these residues account for the low factor IXa activity. Sichler et al. (2003) concluded, that this triple mutant rf9-Y94F/K98T/Y177T (chymotrypsin numbering) probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding.
WO 2010/012451 discloses Factor IX variants with clotting activity in absence of their cofactor and their use for treating bleeding disorders. In WO 2010/012451, the inventors demonstrated that in particular the engineered Factor IX variantITV, containing the mutations V181I, K265T and I383V, is able to bypass factor VIII and correct hemophilic phenotype of F.VIII-knockout mice in the presence of neutralizing antibodies (Milanov et al., 2012).
Hemophilia
The best-known coagulation factor disorders are the hemophilias. Hemophilia is the name of a family of hereditary genetic disorders that impair the body's ability to control blood clotting, or coagulation. Haemophilia A, the most common form, is caused by a mutation of the factor VIII (F.VIII) gene, leading to a deficiency in F.VIII. The inheritance is X-linked recessive; hence, males are affected while females are carriers or very rarely display a mild phenotype. 1 in 5,000 males are affected. Hemophilia B, also known as factor IX (F.IX) deficiency, is the second most common type of hemophilia, but hemophilia B is far less common than hemophilia A.
These genetic deficiencies may lower blood plasma clotting factor levels of coagulation factors needed for a normal clotting process. When a blood vessel is injured, a temporary scab does form, but the missing coagulation factors prevent fibrin formation which is necessary to maintain the blood clot. Thus a haemophiliac does not bleed more intensely than a normal person, but for a much longer amount of time. In severe haemophiliacs even a minor injury could result in blood loss lasting days, weeks, or not ever healing completely. The critical risk here is with normally small bleeds which due to missing F.VIII take long times to heal. In areas such as the brain or inside joints this can be fatal or life debilitating. The bleeding with external injury is normal, but incidence of late re-bleeding and internal bleeding is increased, especially into muscles, joints, or bleeding into closed spaces. Major complications include hemarthrosis, hemorrhage, gastrointestinal bleeding, and menorrhagia.
Though there is no cure for haemophilia, it can be controlled with regular infusions of the deficient clotting factor, i.e. F.VIII in haemophilia A or F.IX in haemophilia B.
In western countries, common standards of care for hemophilia fall into one of two categories: (i) prophylaxis or (ii) on-demand. Prophylaxis involves the infusion of coagulation factor on a regular schedule in order to keep clotting levels sufficiently high to prevent spontaneous bleeding episodes. On-demand treatment involves treating bleeding episodes once they arise.
However, some haemophiliacs develop antibodies (inhibitors) against the replacement factors given to them, so the amount of the factor has to be increased or non-human replacement products must be given, such as porcine F.VIII or modified variants thereof, see e.g. WO 01/68109 A1 (Emory University).
If a patient becomes refractory to replacement coagulation factor as a result of circulating inhibitors, this may be overcome with recombinant human factor VII (NovoSeven®), see also EP 1 282 438 B1 and EP 1 282 439 B1 (Novo Nordisk). A limitation of this approach so far is the short half life of factor VIIa (2 to 3 hours) compared to factor VIII (10 to 14 hours) or factor IX (18 to 30 hours), respectively and depending on the preparation, which makes prophylactic therapy with factor VIIa difficult. Further, the risks of using an already activated protease, like factor VIIa, over prolonged time intervals might carry risks, including thrombotic risks, risks through constant activation of the vascular endothelium and vessel damage, risk of pro-coagulant signalling which could promote tumor growth or metastasis, etc.
WO 02/40544 A2 discloses mutant human factor IX comprising mutations in the heparin binding domain, which decrease the affinity of the mutant human F.IX for heparin compared to wild type F.IX, and their use in the therapeutic intervention of hemophilia B.
Gene Therapy
Hemophilia is ideal for a gene therapeutic approach since the required coagulation is circulating in the blood stream and may therefore be expressed basically everywhere in the body. Further, studies with prophylactic treatment of patients with a severe form of the disease have demonstrated that a minimal elevation of circulating coagulation factor above 1% can already improve the clinical outcome and avoid the majority of lesions caused by the disease, i.e. joint destruction. Several gene therapy approaches have been developed, but testing is still in the early clinical stages. The most promising approaches are currently for the treatment of hemophilia B with adeno-associated viral vectors (AAV).
Intramuscular injection AAV to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX levels. However, dose escalation is not possible in this approach, since the risk of the formation of inhibitory antibodies depends on the amount of F.IX antigen expressed in the muscle per injection site. Estimation in a hemophilia B dog model led to the conclusion, that more than 400 intramuscular injections would be necessary to obtain F.IX expression levels of around 1% in humans (Arruda et al., 2004). This procedure, therefore, is not applicable to humans. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, Schuettrumpf et al. (2005) constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K (F.IX numbering), a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. Besides of the mentioned R338A variant, first described by Chang et al. (1998), another variant, V86A, with higher specific F.IX activity has been described (Chang et al. 2002).
The application of gene therapy strategies for hemophilia A in comparison to hemophilia B is further complicated by the higher immunogenicity and the bigger size of the F.VIII compared to F.IX.
Thus, there is a need in the art for providing further improved means and methods for the treatment and/or prophylaxis of bleeding disorders, in particular hemophilia A and/or B.
Thus, the present invention aims to further improve the methods and means for the treatment and/or prophylaxis of bleeding disorders as present in the prior art and it is, thus, an objective of the present invention to provide further improved methods and means which allow for an effective, specific and targeted treatment and/or prophylaxis of bleeding disorders, in particular hemophilia A and/or B.
Variant Proteins of Factor IX with Clotting Activity in Absence of their Cofactor
According to the present invention this object is solved by providing a variant of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant of F.IX is characterized in that it has clotting activity in absence of its cofactor, wherein the cofactor is factor VIII (F.VIII) or activated factor VIII (F.VIIIa).
The term “variants” as used herein preferably refers to amino acid substitution, addition (insertion) or deletion variants or derivatives of the naturally occurring protein. Variants comprise furthermore an amino acid sequence comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure. Variants can also comprise the substitution or coupling with parts of other molecules or coupling with other molecules.
Amino acid substitutions comprise conservative as well as non conservative replacement by other amino acids or by isosteres (modified amino acids that bear close structural and spatial similarity to protein amino acids), amino acid additions or isostere additions.
Conservative amino acid substitutions typically relate to substitutions among amino acids of the same class. These classes include, for example,                amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine and tyrosine;        amino acids having basic side chains, such as lysine, arginine, and histidine;        amino acids having acidic side chains, such as aspartic acid and glutamic acid; and        amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.        
Factor IX (F.IX) within this patent application refers to the human F.IX protein and cDNA described by Kurachi and Davie, 1982.
Refseq NM_000133 for mRNA/cDNA (SEQ ID NO. 1), and
Refseq NP_000124 for protein sequence (SEQ ID NO. 2).
The amino acid sequence of SEQ ID NO. 2 contains the signal peptide and the pro-peptide of F.IX. The actual numbering starts at −46(Met); +1 is Tyr.
There are several naturally occurring polymorphisms in the gene as well as the amino acid sequence of F.IX. A list from the Hemophilia B Mutation Database, King's College London (see www.kcl.ac.uk/ip/petergreen/haemBdatabase.html) is shown below. For example, the most frequent polymorphism is at position 147 where threonine can be found in 67% and alanine in 33% of the population. Interestingly, the less frequent alanine is present in the only available recombinant F.IX therapeutic.
BaseNameNuc NoChangeAA_ChangeFrequency−1186C→T52%−793G→A44%MseI−698C→T44%BamHI (i)−561T→G 6%25A→GReported once37G→A−44, R→HReported once48A→T−40, I→FReported once181C→Arare192A→G19%353C→Trare709A→Grare1778C→T3/102627T→C6/103747C→A6/103756T→C6/10TaqI (iii)3797C→T6/103905A→T6/10DdeI5505−5076%6550G→CReported once6575C→Gpolymorphic in BrazilPointe-a-Pitre6596G→TReported once(Guadeloupe)XmnI7076G→71%TaqI (ii)9731?Reported once10512A→GReported 5 timesTaqI (i)11111T→65%13275C→T 1/10MspI15625A→G78%17397T→G 1/1020002C→A 4/10MnlI (Malmo)20421G→A147, A→T33%20512T→C178, F→LReported once27731C→G 5/1028364T→Crare29335G→A 1/1029497G→T 1/1029509T→Crare29532C→T 4/1029648G→A 4/1029650A→Grare30134T→C227, V→VReported 7 times30802+AReported 4 times30890C→T257, H→YReported 3 times31012C→T297, N→NReported once31093G→A324, Q→QReported once31103G→A328, V→IReported once32770T→C19%32847T→C“c” allele frequent;“t” allele seen 4 times
Activated factor IX (F.IXa) within this patent application refers to the activated F.IX molecule through cleavage of the 35 amino acid activation peptide as described above.
Since both coagulation factors F.IX and F.VIII always have to be activated before they can exhibit their function both F.IX/F.IXa or F.VIII/F.VIIIa can be used as synonyms.
For the numbering of the amino acid residues the F.IX numbering system is used (according to Kurachi and Davie, 1982, except when indicated otherwise). By some authors in the art, the chymotrypsinogen numbering is used for the description of certain amino acids in homology to the serine protease chymotrypsin. For the present invention the chymotrypsin numbering is only used when explicitly indicated herein.
The “clotting activity” or “functional activity” of F.IX can also be referred to as specific F.IX activity, which is usually measured in Unit/milligram (U/mg). Since one Unit (1 U) of F.IX is referred to as the amount of F.IX in 1 milliliter (ml) of normal human plasma, which corresponds to 5000 ng F.IX, the usual specific activity is around 200 U/mg. Since the specific activity of F.IX is defined as protease activity in the plasma in presence of F.VIII, there is no definition in use in the art for (clotting) activity in absence of cofactor F.VIII. Therefore, the clotting activity in absence of F.VIII, also called “F.VIII-like activity”, is expressed herein as percentage of the activity, which an equal amount of wild type F.IX would exhibit in the presence of F.VIII.
Thus, a F.IX variant has “clotting activity” in absence of its cofactor, when it corrects the blood coagulation deficiency caused by the absence of clotting F.VIII in the blood, which in case of a disease can either be due to absence of the F.VIII protein, presence of a defective F.VIII protein, or inhibition of the F.VIII protein, for example by inhibitory antibodies.
The assay system used in the present invention for determining “clotting activity” or “functional activity” of the variants of a vitamin K-dependent serine protease of the coagulation cascade, preferably of F.IX variants, is an one stage assay based on the aPTT. The activated partial thromboplastin time (aPTT or APTT) is a performance indicator measuring the efficacy of both the “intrinsic” (now referred to as the contact activation pathway) and the common coagulation pathways. Apart from detecting abnormalities in blood clotting, it is also used to monitor the treatment effects with heparin, a major anticoagulant. For the determination of the F.VIII or F.IX activity levels in a sample, the test is performed by spiking the sample into F.VIII or F.IX deficient plasma for measurement of the F.VIII or F.IX activity, respectively. This test is referred to as F.VIII or F.IX one stage assay. Now, F.VIII independent activity of a F.IX variant can be determined by one stage assay and using F.VIII deficient plasma.
Briefly, blood is collected with oxalate or citrate which arrest coagulation by binding calcium. The plasma is separated from the corpuscular parts of the blood by centrifugation. In the case of recombinantly expressed and purified proteins, the protein is diluted in imidazole buffer.
The sample is mixed and added to standardized factor (VIII or IX) deficient plasma. In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the oxalate) are mixed into the plasma sample. The time is measured until a thrombus (clot) forms. The test is termed “partial” due to the absence of tissue factor from the reaction mixture (see Langdell et al., 1953).
Preferably, the variants of factor IX according to the invention have clinical relevant clotting activity (or clotting activity with clinical relevance), i.e. clotting activity which makes the variants suitable for clinical applications, as disclosed herein below.
A preferred clotting activity with clinical relevance is 1% or more clotting activity of the variant in absence of cofactor F.VIII, wherein 100% refers to the activity of wild type F.IX in presence of cofactor F.VIII or F.VIIIa.
Around 1% sustained factor VIII or factor IX levels are enough in prophylactic treatment regimens to prevent major bleeding complications in severe hemophilia patients. To reach a 1% level in a severe hemophilia A patient with a factor IX variant which has “1% F.VIII-like” activity in absence of F.VIII, F.IX variant levels of 100% of normal (around 5000 ng/ml) additional to the already physiologically present F.IX would be necessary. Such a treatment seems feasible and therefore the clinically relevant “factor VIII-like” activity is estimated at 1%.
In an embodiment the variant factor IX of the invention comprises a modification of the 99-loop, preferably by amino acid substitutions, insertions and/or deletions. A modification of the 99-loop can also be achieved by affecting the loop structure by amino acid substitutions, insertions and/or deletions of adjacent amino acid residues or residues interacting otherwise with the 99-loop.
The 99-loop or insertion loop 80-90 (according to chymotrypsinogen numbering) of factor IX encompasses amino acid residues 256 to 268 (F.IX numbering). The 99-loop is positioned near the active site and plays a role in the activation of F.IX. According to Sichler et al. (2003), Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor F.VIIIa. F.X is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.
In WO 2010/012451, the inventors demonstrated that in particular the engineered Factor IX variantITV, containing the mutations V181I, K265T and I383V, is able to bypass factor VIII and correct hemophilic phenotype of F.VIII-knockout mice in the presence of neutralizing antibodies (Milanov et al., 2012). In the present invention, additional modifications are provided to generate even more efficacious F.IX molecules in absence of its cofactor F.VIII as well as to generate potent F.IX variants with increased specific activity in presence of its cofactor F.VIII by combination of different single mutations contributing to an increased activity.
The present invention provides a variant of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant of F.IX is characterized in that it has clotting activity in absence of its cofactor, wherein the cofactor is factor VIII (F.VIII) or activated factor VIII (F.VIIIa).
Said variant factor IX or activated factor IX comprises at least one amino acid substitution in position 265 in combination with amino acid substitution V181I and/or I383V.
The variant with an amino acid substitution in position 265 (preferably K265T or K265A) in combination with amino acid substitution V181I and/or I383V is called the “basis variant factor IX” herein.
Preferably, the variant factor IX according to the invention comprises at least one further amino acid substitution in a position selected from the group of 255 to 269, 383, 181, 6, 44, 72, 75, 102, 105, 122, 185, 224, 263, 338 and/or a modification of the 99-loop.
More preferably, the variant factor IX comprises at least an amino acid substitution selected from K265T, K265A, I383V, V181I, L6F, Q44H, W72R, F75V, S102N, S102K, S102P, S102R, S102Q, S102W, N105S, K122R, E185D, E185S, E185F, E185K, E185P, E185Q, E185R, E224G, E243D, I263S, R338E, T376A and/or a modification of the 99-loop.
The variant factor IX according to the invention comprises at least one amino acid substitution in position 265 in combination with amino acid substitution V181I and/or I383V, and further comprises amino acid substitution(s) in position(s) selected from the group of 6, 11, 25, 44, 54, 72, 75, 78, 86, 89, 102, 105, 113, 119, 122, 125, 135, 139, 154, 159, 185, 195, 196, 211, 219, 222, 224, 236, 243, 251, 260, 262, 263, 268, 289, 299, 302, 304, 310, 319, 330, 334, 336, 338, 366, 368, 376, 383, 386, 391, 392, 394, 399 and/or a modification of the 99-loop,
preferably 255 to 269, 383, 6, 44, 72, 75, 102, 105, 122, 185, 224, 263, 338 and/or a modification of the 99-loop,
more preferably 6, 102 and/or 185.
In a preferred embodiment, the further amino acid substitution(s) is/are selected from the group of
L6F, Q11R, F25L, Q44H, N54D, W72R, F75V, E78D, V86A, N89D, S102N, N105S, E113V, E119V, K122R, E125D, V135A, Q139E, D154N, T159S, E185D, Q195L, V196I, V211I, A219V, C222Y, E224G, I263S, H236L, E243D, I251V, N260K, A262D, I263S, H268D, H268R, C289R, F299T, F302Y, S304F, G310R, K319R, L330I, A334S, R338E, L336H, G366R, P368S, P368I, T376A, I383A, T386A, M391K, K392E, K394R, T399S and/or a modification of the 99-loop,
preferably
L6F, Q44H, W72R, F75V, S102N, S102K, S102P, S102R, S102Q, S102W, N105S, K122R, E185D, E185S, E185F, E185K, E185P, E185Q, E185R, E224G, E243D, I263S, R338E, T376A and/or a modification of the 99-loop,
more preferably L6F, S102N and/or E185D.
In a preferred embodiment, the variant factor IX comprises an amino acid substitution in position 265 (position 98 according to chymotrypsinogen numbering) which is preferably selected from K265T, K265A, K265D, K265E, K265F, K265G, K265H, K265I, K265N, K265S and K265V, more preferably K265T, K265A, in combination with further amino acid substitutions.
In a preferred embodiment, said basis variant factor IX comprises at least an amino acid substitution of the following groups:
Group Aclotting activity in absence of cofactor F.VIII(F.VIII-independent activity)increased compared to wild typeGroup Bclotting activity in absence of cofactor F.VIIIwherein the F.VIII-independent clotting activity is increasedcompared to the respective clotting activity of the basis variant
Amino acid positionAmino acid substitutionGroup A11, 25, 44, 75, 78,Q11R, F25L, Q44H, F75V, E78D, N89D,89, 105, 113, 154,N105S, E113V, D154N, T159S, E185D,159, 185, 211, 224,V211I, E224G, I263S, F302Y, G310R,263, 302, 310, 336L336HGroup B6, 72, 102, 122,L6F, W72R, S102N or other amino acid185, 338, 376substitution (preferably selected fromS102K, S102P, S102R, S102Q, S102W),K122R, E185D or other amino acidsubstitution (preferably selected fromE185S, E185F, E185K, E185P, E185Q,E185R), R338E, T376A
The amino acid substitution in position 265 is preferably selected from K265T, K265A, K265D, K265E, K265F, K265G, K265H, K265I, K265N, K265S and K265V, preferably K265T, K265A.
In a preferred embodiment, the further amino acid substitution(s) is/are selected from position(s) 6, 102 and 185,
preferably,
L6F, S102N, S102K, S102P, S102R, S102Q, S102W, E185D, E185S, E185F, E185K, E185P, E185Q, E185R,
wherein in one embodiment, the amino acid substitution in position 265 is K265T or K265A.
The variant factor IX according to the invention is preferably selected from                variant V181I/K265T/I383V/L6F,        variant V181I/K265T/I383V/S102N,        variant V181I/K265T/I383V/E185D,        variant V181I/K265T/I383V/E185S,        variant V181I/K265T/I383V/L6F/S102N,        variant V181I/K265T/I383V/L6F/S102K,        variant V181I/K265T/I383V/S102N/E185D, and        variant V181I/K265T/I383V/S102N/E185S.        
The variant factor IX according to the invention is preferably selected from                variant V181I/K265A/I383V/L6F,        variant V181I/K265A/I383V/S102N,        variant V181I/K265A/I383V/E185D,        variant V181I/K265A/I383V/E185S,        variant V181I/K265A/I383V/L6F/S102N,        variant V181I/K265A/I383V/L6F/S102K,        variant V181I/K265A/I383V/S102N/E185D, and        variant V181I/K265A/I383V/S102N/E185S.        
More preferably, the variant factor IX according to the invention is selected from                variant V181I/K265A/I383V/L6F, and        variant V181I/K265A/I383V/E185D.Variant Proteins of Factor IX with Increased F.IX Clotting Activity        
According to the present invention this object is solved by providing a variant of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant of F.IX is characterized in that it has increased clotting activity in presence of its cofactor compared to wild type,
wherein the cofactor is factor VIII (F.VIII) or activated factor VIII (F.VIIIa).
The term “increased clotting activity in presence of its cofactor” is also called herein as “hyperfunctional F.IX activity”.
The variant factor IX according to the invention comprises preferably an amino acid substitution in a position selected from the group of
5, 6, 10, 11, 44, 72, 75, 78, 102, 105, 122, 135, 159, 185, 186, 211, 224, 243, 262, 263, 268, 327, 338, 367, 368, 376, 383, 394,
preferably the amino acid substitution R338L or R338E in combination with at least one of K5A, K5F, L6F, V10K, V10F, V10R, Q11R, Q11H, Q11K, Q44H, W72R, F75V, E78D, S102N, N105S, K122R, V135A, T159S, E185D, D186E, V211I, E224G, E243D, A262D, I263S, H268R, R327S, N367D, P368I, T376A, I383A, K394R,
more preferably K5A, L6F, Q11R, Q44H, W72R, F75V, E78D, S102N, N105S, K122R, E185D, D186E, V211I, E224G, E243D, I263S, T376A, K394R.
Preferably, the variant factor IX according to the invention comprises at least one amino acid substitution in a position selected from the group of
5, 6, 10, 11, 44, 72, 75, 102, 105, 122, 185, 224, 243, 263, 338, 376.
The present invention provides a variant of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant of F.IX is characterized in that it has increased clotting activity in presence of its cofactor compared to wild type, wherein the cofactor is factor VIII (F.VIII) or activated factor VIII (F.VIIIa).
Said variant factor IX or activated factor IX comprises at least one amino acid substitution in position 338 (preferably R338L or R338E) in combination with an amino acid substitution in position 377 (preferably S377W).
In a preferred embodiment, said variant factor IX comprising an amino acid substitution in position 338 (preferably R338L or R338E) in combination with an amino acid substitution in position 377 (preferably S377W), comprises at least an amino acid substitution of the following groups:
Group Dincreased clotting activity in presence of cofactor F.VIII(F.VIII-dependent activity)increased compared to wild type
Amino acid positionAmino acid substitutionGroup D5, 6, 10, 11, 44, 72,K5A, L6F, Q11R, Q44H, W72R, F75V,75, 102, 105, 122,E78D, S102N, N105S, K122R, E185D,185, 211, 224, 243,D186E, V211I, E224G, E243D, I263S,263, 338, 376R338E, T376A, K394R
The present invention provides a variant of factor IX (F.IX), which is characterized in that it has increased clotting activity in presence of its cofactor compared to wild type,
wherein the cofactor is factor VIII (F.VIII) or activated factor VIII (F.VIIIa),
said variant factor IX or activated factor IX comprising an amino acid substitution in position 338 in combination with amino acid substitution(s) in position(s) selected from the group of 4, 5, 6, 10, 11, 44, 72, 75, 78, 102, 105, 122, 135, 159, 185, 186, 211, 224, 243, 262, 263, 265, 268, 327, 367, 368, 376, 377, 383, 394,
preferably 4, 5, 6, 10, 11, 44, 72, 75, 102, 105, 122, 185, 224, 243, 263, 265, 376, 377,
more preferably 377, 10, 4, 5 and/or 265.
In a preferred embodiment, the further amino acid substitution(s) is/are selected from the group of
G4Y, K5A, K5F, L6F, V10K, V10F, V10R, Q11R, Q11H, Q11K, Q44H, W72R, F75V, E78D, S102N, N105S, K122R, V135A, T159S, E185D, D186E, V211I, E224G, E243D, A262D, I263S, K265T, H268R, R327S, N367D, P368I, T376A, S377W, I383A, K394R, preferably K5A, L6F, Q11R, Q44H, W72R, F75V, E78D, S102N, N105S, K122R, E185D, D186E, V211I, E224G, E243D, I263S, K265T, T376A, S377W, K394R,
more preferably S377W, V10K, G4Y, K5A and/or K265T.
In a preferred embodiment, the variant factor IX comprises the amino acid substitution in position 338 in combination with                an amino acid substitution in position 5,        an amino acid substitution in position 10, and/or        an amino acid substitution in position 377.        
Preferably, the amino acid substitution in position 338 is R338L or R338E,
wherein the amino acid substitution in position 5 is K5A,
wherein the amino acid substitution in position 10 is V10K,
and/or wherein the amino acid substitution in position 377 is S377W.
The variant factor IX according to the invention is preferably selected from                variant V10K/R338L        variant R338L/S377W        variant V10K/R338L/S377W        variant V10K/R338L/S377W/L6F        variant V10K/R338L/S377W/E243D        variant V10K/R338L/S377W/E224G        variant V10K/R338L/S377W/L6F/E224G        variant V10K/R338L/S377W/E243D/E224G        variant V10K/R338L/S377W/K265T        variant K5A/R338L        variant K5A/R338L/S377W        variant K5A/V10K/R338L/S377W        variant G4Y/V86A/R338L/S377W, and        variant G4Y/V86A/R338L/S377W/K265T.Disclaimer:        
The present invention does not encompass a variant factor IX already disclosed in the earlier application of the inventors WO 2010/012451. In particular the present invention does not encompass:                single variants: R338A, S377W, G4Y, V86A, K265T, K265A        variant G4Y/V10K,        variant S340T/R338A/Y345T,        variant R338A/S377W,        variant S360A/R338A/S377W,        variant V86A/R338A/S377W,        variant G4Y/R338A/S377W,        variant R338A/K265T,        variant K265T/R338A/I383V,        variant Y259F/K265T/R338A/T340S/Y345T,        variant V181I, K265T/I383V,        variant V181I/K265T/R338A/S377W/I383V.        
Furthermore, the present invention does not encompass the following variants, such as disclosed in Chang et al., 1998, WO 99/03496 A1, US 2008/167219 A1, Chang et al., 2002, or Kao et al., 2013 or reviewed in Quade-Lyssy et al., 2012:                single variants: K5A, V10K, V86A, E277A, R338A, R338L,        single variants: S102A, E113A, K122A, N105A,        variant K5A/V10K,        variant V86A/E277A,        variant E277A/R338A,        variant V86A/E277A/R338A,        variant V86A/E277A/R338L,        variant Y259F/K265T/Y345T.        
Conjugates
In a preferred embodiment, the variants of factor IX according to the invention comprise a further compound or moiety, which is preferably covalently attached to the variant (conjugate).
Preferably, the further compound or moiety is selected from                a protein, such as albumin,        a label, such as chromophor, fluorophor, isotope,        
and/or                a bio-/polymer, such as chitosan, PEG.        
In one embodiment, the chitosan-conjugates are suitable for gene therapy, such as for oral gene delivery of the F.IX variants.
Nucleic Acids of the F.IX Variants and Pharmaceutical Compositions
According to the present invention the above object is furthermore solved by providing nucleic acids encoding the variant factor IX according to the present invention.
A “nucleic acid” refers to DNA, RNA and derivatives thereof, DNA and/or RNA comprising modified nucleotides/nucleosides.
Preferably, the nucleic acid is operably linked to a promoter and/or terminator sequence. Preferred promoter and/or terminator sequences are the human alpha1 anti-trypsin promoter, the hepatic locus control region 1, or the cytomegalovirus promoter and a polyadenylation signal of human or bovine growth hormone of the Simianese Virus 40.
The skilled artisan is able to select suitable promoter and/or terminator sequences.
A nucleic acid is “operably linked” to a promoter and/or terminator sequence when the transcription/translation of the nucleic acid is controlled by the respective promoter/terminator, preferably in a cell and by the cellular transcription/translation machinery, such that e.g. the encoded protein can be obtained from the nucleic acid.
Preferably, the nucleic acid is an expression plasmid, a gene therapy or delivery construct, a sequence encoded in a gene transfer vector, a gene sequence used for DNA modification or repair, or similar.
Preferred gene therapy or delivery constructs are viral and non-viral vectors, such as adeno-associated viral vectors (AAV), plasmid vectors, or minicircle vectors, as described e.g. in Schuettrumpf et al., 2005 and Milanov et al., 2012, or chitosan nanoparticles.
A preferred gene therapy or delivery construct is a minicircle vector, such as a minicircle under the control of a liver-directed or a CMV promoter.
For example, such as for in vivo expression, a nucleic acid encoding the variant F.IX according to the present invention (e.g. a F.IX expression cassette) is introduced into a minicircle producer plasmid, such as pMC.BESPX-MCS2 (System Biosciences), and controlled by e.g. the strong liver-specific enhancer/promoter HCR/hAAT (hepatic locus control region 1/human α-1-antitrypsin) or the CMV promoter.
Such minicircle vectors can be administered by injection, parenterally, or orally when encapsulated in chitosan nanoparticles.
For details, see Example 1.
A preferred gene therapy or delivery construct are chitosan nanoparticles, which are particularly suitable for oral gene delivery and are described in the art, see Mao et al., 2001 or Bowman & Leong, 2006.
The nanoparticles contain chitosan and the nucleic acid, preferably DNA.
Chitosan, a non-toxic and biodegradable polysaccharide derived from partial deacetylation of chitin, can be used to form nanoparticles as a potent oral drug and gene delivery system, reviewed in Bowman & Leong, 2006. Nanoparticles are formed through electrostatic interaction between anionic DNA and cationic chitosan protecting the encapsulated DNA from digestion and enhancing uptake in the gut by improving intestinal trans- and paracellular permeability and its mucoadhesive nature.
For details, see Example 2
According to the present invention the object is furthermore solved by providing a pharmaceutical composition comprising at least one variant of factor IX (F.IX) of the invention or at least one nucleic acid of the invention, and optionally pharmaceutically acceptable carrier(s) and/or excipient(s).
Suitable pharmaceutically acceptable carrier(s) and/or excipient(s) are known in the art. The skilled artisan will selected the preferred pharmaceutically acceptable carrier(s) and/or excipient(s) depending on the intended application of the pharmaceutical composition, such as disorder to be treated, patient to be treated, treatment regimen etc.
Medical Uses
According to the present invention the object is furthermore solved by providing the variants of factor IX, as disclosed in the present invention or the nucleic acids encoding them or the pharmaceutical compositions of the invention for the diagnosis, prevention and/or treatment of diseases.
The disease to be diagnosed, prevented and or treated is preferably a bleeding disorder or bleeding.
A “bleeding disorder” is preferably hemophilia A and/or hemophilia B, hemophilia caused or complicated by inhibitory antibodies to factor VIII, by a deficiency of factor VIII or factor IX, or by the presence of a non functional factor VIII or factor IX protein, or any other bleeding or bleeding tendency.
Preferably, the bleeding disorder is hemophilia A, hemophilia caused or complicated by inhibitory antibodies to factor F.VIII or F.VIIIa, hemophilia B.
Preferably, the bleeding disorder or bleeding is a bleeding disorder where by-passing agents are used, including e.g. neonatal coagulopathies; severe hepatic disease; high-risk surgical procedures; traumatic blood loss; bone marrow transplantation; thrombocytopenias and platelet function disorders; urgent reversal of oral anticoagulation; congenital deficiencies of factors V, VII, X, and XI; and von Willebrand disease with inhibitors to von Willebrand factor, blood loss in connection with large injuries, cerebral bleedings, thrombocyte function disorders.
Preferably, the variant of factor IX (F.IX), the nucleic acid or the pharmaceutical composition of the invention are used for protein infusion therapy, cellular therapy, gene delivery or therapy and/or prophylaxis of a bleeding disorder or bleeding.
In one embodiment, gene therapy or delivery comprises the use of a gene therapy or delivery construct that comprises the variant of factor IX (F.IX), the nucleic acid or the pharmaceutical composition.
Preferred gene therapy or delivery constructs are viral and non-viral vectors, such as adeno-associated viral vectors (AAV), plasmid vectors, or minicircle vectors, as described e.g. in Schuettrumpf et al., 2005 and Milanov et al., 2012, or chitosan nanoparticles.
Such minicircle vectors can be administered by injection, parenterally, or orally.
The variants of the present invention are suitable tools to treat patients with bleeding disorder using protein administration, or cell- or gene therapeutic administration (viral, DNA, RNA, or other vectors). Diseases for treatment are hemophilia A and B, also caused or complicated by inhibitory antibodies to FVIII, for treatment of bleeding and for prophylactic therapy.
The inventors have shown that the variants of the present invention                improve thrombin generation dose-dependent in the absence of F.VIII and show marginal effects in the presence of F.VIII in comparison to wild type F.IX,        improve F.X activation in the presence and absence of F.VIII,        correct clotting time in presence of inhibitory antibodies against F.VIII (confirming the function of the tested F.IX variants also in presence of F.VIII inhibitors),        are not pre-activated but zymogen-like,        correct coagulation and stop bleeding in vivo (being the first evidence that F.IX variants can serve as hemostatically active therapeutics in vivo),        
For further details, see Examples 6-8 and FIGS. 1-7.
Screening Method
According to the present invention, the object is furthermore solved by providing a method for screening of anticoagulant compounds (anticoagulants), preferably substances that directly inhibit F.IXa.
Such a method comprises the use of at least one variant factor IX of the present invention, as defined herein.
In such a method, no further components of the tenase complex are necessary (wherein “tenase” refers to complex of the activated forms of the blood coagulation factors factor VIII (F.VIIIa) and factor IX (F.IXa). It forms on a phospholipid surface in the presence of calcium and is responsible for the activation of factor X).
An advantageous aspect of the described variants is, that without need for F.VIIIa and higher activity towards both, F.X cleavage and chromogenic substrate cleavage, the variants are suitable tools in diagnostic testing systems or for the development and screening for direct F.IXa inhibitory substances, which since a long time are desired as anticoagulants, but for which no effective screening was possible, due to the low efficacy of F.IXa without assembly in the tenase complex.
A screening method according to the invention is preferably a method for identifying a compound which binds to a variant factor IX of the present invention and/or which modulates its activity,
preferably comprising the following steps:                providing compounds/substances to be tested,        providing at least one variant factor IX of the present invention,        contacting a compound/substance to be tested with the at least one variant factor IX of the present invention,        determining whether the compound/substance binds to the at least one variant factor IX,        optionally, determining whether the compound/substance modulates the activity of the at least one variant factor IX.        
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
Shown are means±SEM. **p<0.01 according to ANOVA using Dunnett's test for multiple comparison with the HB group.